ProSAAS-Derived Peptides are Colocalized with Neuropeptide Y and Function as Neuropeptides in the Regulation of Food Intake Jonathan H. Wardman 1 , Iryna Berezniuk 1,2 , Shi Di 3 , Jeffrey G. Tasker 3,4 , Lloyd D. Fricker 1,2 * 1 Department of Molecular Pharmacology, Albert Einstein College of Medicine, New York, New York, United States of America, 2 Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, New York, New York, United States of America, 3 Department of Cell and Molecular Biology, Tulane University, New Orleans, Louisiana, United States of America, 4 Neuroscience Program, Tulane University, New Orleans, Louisiana, United States of America Abstract ProSAAS is the precursor of a number of peptides that have been proposed to function as neuropeptides. Because proSAAS mRNA is highly expressed in the arcuate nucleus of the hypothalamus, we examined the cellular localization of several proSAAS-derived peptides in the mouse hypothalamus and found that they generally colocalized with neuropeptide Y (NPY), but not a-melanocyte stimulating hormone. However, unlike proNPY mRNA, which is upregulated by food deprivation in the mediobasal hypothalamus, neither proSAAS mRNA nor proSAAS-derived peptides were significantly altered by 1–2 days of food deprivation in wild-type mice. Furthermore, while proSAAS mRNA levels in the mediobasal hypothalamus were significantly lower in Cpe fat/fat mice as compared to wild-type littermates, proNPY mRNA levels in the mediobasal hypothalamus and in other subregions of the hypothalamus were not significantly different between wild-type and Cpe fat/fat mice. Intracerebroventricular injections of antibodies to two proSAAS-derived peptides (big LEN and PEN) significantly reduced food intake in fasted mice, while injections of antibodies to two other proSAAS-derived peptides (little LEN and little SAAS) did not. Whole-cell patch clamp recordings of parvocellular neurons in the hypothalamic paraventricular nucleus, a target of arcuate NPY projections, showed that big LEN produced a rapid and reversible inhibition of synaptic glutamate release that was spike independent and abolished by blocking postsynaptic G protein activity, suggesting the involvement of a postsynaptic G protein-coupled receptor and the release of a retrograde synaptic messenger. Taken together with previous studies, these findings support a role for proSAAS-derived peptides such as big LEN as neuropeptides regulating food intake. Citation: Wardman JH, Berezniuk I, Di S, Tasker JG, Fricker LD (2011) ProSAAS-Derived Peptides are Colocalized with Neuropeptide Y and Function as Neuropeptides in the Regulation of Food Intake. PLoS ONE 6(12): e28152. doi:10.1371/journal.pone.0028152 Editor: Silvana Gaetani, Sapienza University of Rome, Italy Received July 12, 2011; Accepted November 2, 2011; Published December 2, 2011 Copyright: ß 2011 Wardman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported primarily by National Institutes of Health grants DA-04494 (to LDF) and MH066958 (to JGT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lloyd.fricker@einstein.yu.edu Introduction Peptides play major roles in diverse physiological functions, including as hormones, neurotransmitters, and growth factors [1,2]. Bioactive peptides are typically produced by cleavage of larger precursor proteins at specific sites containing basic residues, often in pairs such as Arg-Arg or Lys-Arg [3,4]. Endopeptidases such as prohormone convertase (PC) 1/3 and 2 cut the precursors at these sites, generating intermediate peptides with C-terminal basic amino acid extensions [5,6]. Carboxypeptidases then trim these basic residues from the ends of the peptides, after which the peptides may need further modification (such as amidation) before the peptides are biologically active [7]. Carboxypeptidase E (CPE) is the major peptide-processing carboxypeptidase; this enzyme is found in all neuroendocrine tissues and cleaves many C-terminally extended peptides to generate the mature bioactive form [8]. The fat mutation, a spontaneous mouse mutation, was found to be a point mutation in CPE that causes the peptidase to be inactive [9]. Mice homozygous for this mutation, which was renamed Cpe fat , show an accumulation of the peptide processing intermediates containing basic residues on their C-termini and a reduction in levels of fully processed peptides [10]. An affinity chromatography technique was used to isolate these neuropeptide intermediates after which the peptides were sequenced using tandem mass spectrometry [11]. This approach identified the processing intermediates of many previously characterized neuroendocrine peptides as well as several novel peptides. Five of these novel peptides were named based on primary amino acid sequences contained within the larger peptides: big SAAS, little SAAS, PEN, big LEN, and little LEN. All five of these peptides were derived from the same precursor, named proSAAS, by cleavage at basic amino acid-containing sites (Figure 1). The involvement of CPE in the biosynthesis of proSAAS-derived peptides indicates that these peptides are processed in the regulated secretory pathway of neuroendocrine cells. Although there is no sequence similarity among proSAAS and other proteins, there are some features in common between proSAAS and 7B2, a member of the granin family [12]. Both proteins are broadly expressed in brain and neuroendocrine tissues [13], are generally the same size, and contain multiple cleavage sites for the PCs and CPE [11,12,14]. 7B2 is an inhibitor of PC2 [15,16]. Similar studies with proSAAS showed that a small region PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e28152